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Functional Analysis of Tropomyosin Isoforms in Vivo

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Functional Analysis of Tropomyosin Isoforms in Vivo FUNCTIONAL ANALYSIS OF TROPOMYOSIN ISOFORMS IN VIVO by Aeri Cho A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland March, 2015 Abstract Precise regulation of a dynamic actin cytoskeleton is an essential function of animal cells without which vesicle trafficking, cytokinesis, cell adhesion and cell movement would be impossible. Therefore, understanding the mechanisms underlying actin dynamics is fundamental for understanding basic cellular biology as well as gaining insight into mechanisms of embryonic development, tissue homeostasis and regeneration, and pathological processes such as inflammation and tumor metastasis. Actin filaments are a major source of the protrusive and contractile forces that drive many cellular behaviors. Contractile forces require the action of non-muscle myosin II, which assembles onto actin filaments to form acto-myosin. The interaction between actin and myosin can occur spontaneously in vitro, but in cells it is regulated by accessory proteins including Tropomyosins (Tms). A complete understanding of Tm function has been elusive due in part to the large number of isoforms: 44 predicted isoforms from 4 genes in humans. The goal of this study was to decipher the functional roles of different Tm isoforms at the molecular, cellular and tissue levels in vivo. Drosophila egg chambers are a genetically tractable system that expresses far fewer Tm isoforms than mammalian cells. We identified three tropomyosin isoforms expressed in follicle cells, including one previously annotated as muscle-specific. We generated and characterized isoform- specific antibodies, RNAi lines, and mutant alleles, and discovered that they function non-redundantly in the two cell types we studied: border cells, a well-studied example of collective migration, and epithelial follicle cells, which develop contractile stress fibers that shape the egg chamber. ii We also found that Tm mutations interact genetically with Psidin, a conserved but poorly characterized protein that we found regulates lamellipodia dynamics in both Drosophila and mammalian cells. Although strong loss-of-function mutations in either Tm or Psidin inhibit border cell migration individually, mutation of Tm suppresses the Psidin mutant phenotype. Furthermore, in a biochemical assay, we showed that Psidin inhibited Tm protein binding to actin filaments. Taken together this work has generated reagents that will be generally useful in deciphering Tm isoform functions in vivo in Drosophila, has revealed isoform-specific functions, and has revealed a novel genetic and biochemical interaction. Advisor: Dr. Denise J. Montell Molecular, Cellular and Developmental Biology, UCSB Reader: Dr. Douglas Robinson Department of Cell Biology, Johns Hopkins School of Medicine iii Acknowledgments First and foremost, I would like to thank my advisor, Dr. Denise Montell. One of the best decisions I made in my life was to join her lab. Her original ideas and insights always fascinated me and made me wonder about science. She not only advised me to go forward with my project during hardships, but she also taught me to become more independent and more adventurous. I cannot thank enough for her decision to move to University of California, Santa Barbara and providing us with wonderful working environment. I am surely going to miss walks over to the beach and the beautiful weather. I would like to thank past and present lab members of the Montell lab. Especially Adi Gurav for helping me out whenever I reached out for help. Also for enjoyable coffee breaks and moments of venting out when experiments did not work. Special thanks to Hsiang Chen and Leanna Li for delightful brunch conversations. I would also like to thank my undergraduate assistant, Tess Whitwam for her tremendous help and her commitment to lab work despite busy class schedule. I would like to thank my thesis committee members Dr. Douglas Robinson, Dr. Susan Craig, and Dr. Deborah Andrew for their time and valuable advice on my project. Special thanks to Dr. Douglas Robinson, for reading my thesis and providing me lots of insights regarding my favorite protein, tropomyosin. I would also like to thank parents for their endless love and support. They were always on my side and encouraged me to do whatever I pursued to do. I am happy that I could go back to Korea after 13 years of study in the states to spend the good times that I iv have missed. Also big thanks to my brother and his family. Without my family’s support, this dissertation would not have been possible. Last but not least, I would like to thank my husband Jeonggil Ko. Although we have been apart longer than we have been together, there wasn’t a single moment when I felt lack of attention or love. I thank him for being patient with me and for always encouraging me at times when I was discouraged with my research. Without him, I would not have made it to the end. We have been counting down the days till we meet and I am looking forward to the wonderful journey ahead of us. v Table of Contents Abstract ......................................................................................................................... ii Acknowledgements ....................................................................................................... iv Table of contents .......................................................................................................... vi List of Figures ............................................................................................................... ix List of Tables ................................................................................................................ xi Chapter 1: Introduction ................................................................................................ 1 Overview ........................................................................................................................ 2 I. Actin cytoskeleton ...................................................................................................... 3 Different actin structures and cell migration ............................................................ 3 Different actin proteins and their functions .............................................................. 4 II. Drosophila egg chamber as a model system ............................................................ 6 Border cell migration .............................................................................................. 6 Epithelial follicle cells – organization and regulation ............................................... 8 How tissues shape their form and structure – egg chamber rotation and elongation 10 Thesis overview............................................................................................................ 11 Reference ..................................................................................................................... 12 Chapter 2: Functional analysis of Tropomyosin isoforms in vivo ............................. 26 Introduction ................................................................................................................. 27 Tropomyosin in mammalian system ...................................................................... 27 Non-muscle Tropomyosin as a regulator of actin cytoskeleton ............................... 28 Importance of studying Tropomyosin regulation ................................................... 29 vi Drosophila Tropomyosin ....................................................................................... 30 Results .......................................................................................................................... 32 Three Tm1 isoforms are expressed in the Drosophila egg chamber ....................... 32 Analysis of different Tm1 isoforms in the egg chambers using isoform specific antibodies ............................................................................................................. 33 Tm1 is highly expressed on the actin filaments in the basal layer of the epithelial follicle cells .......................................................................................................... 35 Tm1 is required for border cell migration .............................................................. 36 Three Tm1 isoforms have non-redundant functions in border cell migration .......... 37 Tm1 functions to regulate actin filaments in the basal layer of the epithelial follicle cells ....................................................................................................................... 40 Three Tm1 isoforms function non-redundantly to regulate actin filaments in the basal layer of the epithelial follicle cells ................................................................. 41 Tm1 isoforms interact with each other to regulate their function and expression ... 42 Discussion .................................................................................................................... 44 Tm1-L is expressed in non-muscle cells ................................................................. 45 Tm1-I may play a role in oskar mRNA localization .............................................. 46 Tropomyosin impacts actin dynamics ................................................................... 48 Material and Methods ................................................................................................
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